Methods for treating Alzheimer&#39;s disease using hydroxyethylene compounds containing a heterocyclic amide bond isostere

ABSTRACT

Disclosed are methods for treating Alzheimer&#39;s disease, and other diseases, and/or inhibiting beta-secretase enzyme, and/or inhibiting deposition of A beta peptide in a mammal, by use of compounds of formula (I) wherein X, R 1 , and R 2  are defined herein.

This application claims priority to U.S. Provisional Patent Application60/392,129, filed Jun. 27, 2002.

FIELD OF THE INVENTION

The present invention relates to the treatment of Alzheimer's diseaseand other similar diseases, and more specifically to the use ofcompounds that inhibit beta-secretase, an enzyme that cleaves amyloidprecursor protein to produce A beta peptide, a major component of theamyloid plaques found in the brains of Alzheimer's sufferers, in suchmethods.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive degenerative disease of thebrain primarily associated with aging. Clinical presentation of AD ischaracterized by loss of memory, cognition, reasoning, judgment, andorientation. As the disease progresses, motor, sensory, and linguisticabilities are also affected until there is global impairment of multiplecognitive functions. These cognitive losses occur gradually, buttypically lead to severe impairment and eventual death in the range offour to twelve years.

Alzheimer's disease is characterized by two major pathologicobservations in the brain: neurofibrillary tangles and beta amyloid (orneuritic) plaques, comprised predominantly of an aggregate of a peptidefragment know as A beta. Individuals with AD exhibit characteristicbeta-amyloid deposits in the brain (beta amyloid plaques) and incerebral blood vessels (beta amyloid angiopathy) as well asneurofibrillary tangles. Neurofibrillary tangles occur not only inAlzheimer's disease but also in other dementia-inducing disorders. Onautopsy, large numbers of these lesions are generally found in areas ofthe human brain important for memory and cognition.

Smaller numbers of these lesions in a more restricted anatomicaldistribution are found in the brains of most aged humans who do not haveclinical AD. Amyloidogenic plaques and vascular amyloid angiopathy alsocharacterize the brains of individuals with Trisomy 21 (Down'sSyndrome), Hereditary Cerebral Hemorrhage with Amyloidosis of theDutch-Type (HCHWA-D), and other neurodegenerative disorders.Beta-amyloid is a defining feature of AD, now believed to be a causativeprecursor or factor in the development of disease. Deposition of A betain areas of the brain responsible for cognitive activities is a majorfactor in the development of AD. Beta-amyloid plaques are predominantlycomposed of amyloid beta peptide (A beta, also sometimes designatedbetaA4). A beta peptide is derived by proteolysis of the amyloidprecursor protein (APP) and is comprised of 39-42 amino acids. Severalproteases called secretases are involved in the processing of APP.

Cleavage of APP at the N-terminus of the A beta peptide bybeta-secretase and at the C-terminus by one or more gamma-secretasesconstitutes the beta-amyloidogenic pathway, i.e. the pathway by which Abeta is formed. Cleavage of APP by alpha-secretase produces alpha-sAPP,a secreted form of APP that does not result in beta-amyloid plaqueformation. This alternate pathway precludes the formation of A betapeptide. A description of the proteolytic processing fragments of APP isfound, for example, in U.S. Pat. Nos. 5,441,870; 5,721,130; and5,942,400.

An aspartyl protease has been identified as the enzyme responsible forprocessing of APP at the beta-secretase cleavage site. Thebeta-secretase enzyme has been disclosed using varied nomenclature,including BACE, Asp, and Memapsin. See, for example, Sindha et al.,1999, Nature 402:537-554 (p501) and published PCT applicationWO00/17369.

Several lines of evidence indicate that progressive cerebral depositionof beta-amyloid peptide (A beta) plays a seminal role in thepathogenesis of AD and can precede cognitive symptoms by years ordecades. See, for example, Selkoe, 1991, Neuron 6:487. Release of A betafrom neuronal cells grown in culture and the presence of A beta incerebrospinal fluid (CSF) of both normal individuals and AD subjects hasbeen demonstrated. See, for example, Seubert et al., 1992, Nature359:325-327.

It has been proposed that A beta peptide accumulates as a result of APPprocessing by beta-secretase, thus inhibition of this enzyme's activityis desirable for the treatment of AD. In vivo processing of APP at thebeta-secretase cleavage site is thought to be a rate-limiting step in Abeta production, and is thus a therapeutic target for the treatment ofAD. See for example, Sabbagh, M., et al., 1997, Alz. Dis. Rev. 3, 1-19.

BACE1 knockout mice fail to produce A beta, and present a normalphenotype. When crossed with transgenic mice that over express APP, theprogeny show reduced amounts of A beta in brain extracts as comparedwith control animals (Luo et al., 2001 Nature Neuroscience 4:231-232).This evidence further supports the proposal that inhibition ofbeta-secretase activity and reduction of A beta in the brain provides atherapeutic method for the treatment of AD and other beta amyloiddisorders.

At present there are no effective treatments for halting, preventing, orreversing the progression of Alzheimer's disease. Therefore, there is anurgent need for pharmaceutical agents capable of slowing the progressionof Alzheimer's disease and/or preventing it in the first place.

Compounds that are effective inhibitors of beta-secretase, that inhibitbeta-secretase-mediated cleavage of APP, that are effective inhibitorsof A beta production, and/or are effective to reduce amyloid betadeposits or plaques, are needed for the treatment and prevention ofdisease characterized by amyloid beta deposits or plaques, such as AD.

Thompson, et al., (Thompson, S. K., et al., J. Med. Chem., 1994;37:3100-3107) discloses compounds of formula (I)

wherein R₁ is

-   -   a) Cbz-Val;    -   b) Cbz-Ala;    -   c) Cbz; or    -   d) Boc;        R₂ is    -   a) H;    -   b) C₁₋₆ alkyl, alkenyl, or alkynyl; or    -   c) cycloalkyl; and        X is    -   a) C═O; or    -   b) (CH₂)OH.

Thompson, et al. discloses how to make the above compounds, well as theinhibitory activity of these compounds against HIV-1 protease. Thedisclosure of Thompson, et al. is incorporated rein by reference in itsentirety.

SUMMARY OF INVENTION

The present invention relates to methods of treating a subject who has,or in preventing a subject from developing, a disease or conditionselected from the group consisting of Alzheimer's disease, for helpingprevent or delay the onset of Alzheimer's disease, for helping to slowthe progression of Alzheimer's disease, for treating subjects with mildcognitive impairment (MCI) and preventing or delaying the onset ofAlzheimer's disease in those who would progress from MCI to AD, fortreating Down's syndrome, for treating humans who have HereditaryCerebral Hemorrhage with Amyloidosis of the Dutch-Type, for treatingcerebral amyloid angiopathy and preventing its potential consequences,i.e. single and recurrent lobar hemorrhages, for treating otherdegenerative dementias, including dementias of mixed vascular anddegenerative origin, dementia associated with Parkinson's disease,frontotemporal dementias with parkinsonism (FTDP), dementia associatedwith progressive supranuclear palsy, dementia associated with corticalbasal degeneration, or diffuse Lewy body type of Alzheimer's disease andwho is in need of such treatment which comprises administration of atherapeutically effective amount of a compound of formula (I):

wherein R₁ is

-   -   a) Cbz-Val;    -   b) Cbz-Ala;    -   c) Cbz; or    -   d) Boc;        R₂ is    -   a) H;    -   b) C₁₋₆ alkyl, alkenyl, or alkynyl; or    -   c) cycloalkyl; and        X is    -   a) C═O; or    -   b) (CH₂)OH;        or pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to methods of treating asubject who has, or in preventing a subject from developing, a diseaseor condition selected from the group consisting of Alzheimer's disease,for helping prevent or delay the onset of Alzheimer's disease, forhelping to slow the progression of Alzheimer's disease, for treatingsubjects with mild cognitive impairment (MCI) and preventing or delayingthe onset of Alzheimer's disease in those who would progress from MCI toAD, for treating Down's syndrome, for treating humans who haveHereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type, fortreating cerebral amyloid angiopathy and preventing its potentialconsequences, i.e. single and recurrent lobar hemorrhages, for treatingother degenerative dementias, including dementias of mixed vascular anddegenerative origin, dementia associated with Parkinson's disease,frontotemporal dementias with parkinsonism (FTDP), dementia associatedwith progressive supranuclear palsy, dementia associated with corticalbasal degeneration, or diffuse Lewy body type of Alzheimer's disease andwho is in need of such treatment which comprises administration of atherapeutically effective amount of a compound of formula (I):

wherein R₁ is

-   -   a) Cbz-Val;    -   b) Cbz-Ala;    -   c) Cbz; or    -   d) Boc;        R₂ is    -   a) H;    -   b) C₁₋₆ alkyl, alkenyl, or alkynyl; or    -   c) cycloalkyl; and        X is    -   a) C═O; or    -   b) (CH₂)OH;        or pharmaceutically acceptable salt thereof.

In a preferred embodiment the methods comprise administration of acompound of the formula II:

wherein R₁ is Cbz-Val or Boc;

-   -   R₂ is H, methyl, ethyl, n-propyl, isopropyl, or C(CH₃)₂—CHCH₂;        and    -   X is C═O or (CH₂)OH;    -   or pharmaceutically acceptable salt thereof.

The compounds useful in the methods of the present invention, may haveasymmetric centers and occur as racemates, racemic mixtures and asindividual diastereomers, or enantiomers with all isomeric forms beingincluded in the present invention.

When any variable occurs more than one time in any constituent or inFormula I, its definition on each occurrence is independent of itsdefinition at every other occurrence. Also, combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

By “alkyl” and “C₁-C₆ alkyl” in the present invention is meant straightor branched chain alkyl groups having 1-6 carbon atoms, such as, methyl,ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl,2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and3-methylpentyl. It is understood that in cases where an alkyl chain of asubstituent (e.g. of an alkyl, alkoxy or alkenyl group) is shorter orlonger than 6 carbons, it will be so indicated in the second “C” as, forexample, “C₁-C₁₀” indicates a maximum of 10 carbons.

“Alkenyl” and “C₂-C₆ alkenyl” means straight and branched hydrocarbonradicals having from 2 to 6 carbon atoms and from one to three doublebonds and includes, for example, ethenyl, propenyl, 1-but-3-enyl,1-pent-3-enyl, 1-hex-5-enyl, and the like.

“Alkynyl” and “C₂-C₆ alkynyl” means straight and branched hydrocarbonradicals having from 2 to 6 carbon atoms and one or two triple bonds andincludes ethynyl, propynyl, butynyl, pentyn-2-yl and the like.

As used herein, the term “cycloalkyl” refers to saturated carbocyclicradicals having three to twelve carbon atoms. The cycloalkyl can bemonocyclic, or a polycyclic fused system. Examples of such radicalsinclude cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Preferredcycloalkyl groups are cyclopentyl, cyclohexyl, and cycloheptyl. Thecycloalkyl groups herein are unsubstituted or, as specified, substitutedin one or more substitutable positions with various groups. For example,such cycloalkyl groups may be optionally substituted with, for example,C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, hydroxy, cyano, nitro, amino,mono(C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, C₂-C₆alkenyl, C₂-C₆alkynyl,C₁-C₆ haloalkyl, C₁-C₆ haloalkoxy, amino(C₁-C₆)alkyl,mono(C₁-C₆)alkylamino(C₁-C₆)alkyl or di(C₁-C₆)alkylamino(C₁-C₆)alkyl.

In one aspect, this method of treatment can be used where the disease isAlzheimer's disease.

In another aspect, this method of treatment can help prevent or delaythe onset of Alzheimer's disease.

In another aspect, this method of treatment can help slow theprogression of Alzheimer's disease.

In another aspect, this method of treatment can be used where thedisease is mild cognitive impairment.

In another aspect, this method of treatment can be used where thedisease is Down's syndrome.

In another aspect, this method of treatment can be used where thedisease is Hereditary Cerebral Hemorrhage with Amyloidosis of theDutch-Type.

In another aspect, this method of treatment can be used where thedisease is cerebral amyloid angiopathy.

In another aspect, this method of treatment can be used where thedisease is degenerative dementias.

In another aspect, this method of treatment can be used where thedisease is diffuse Lewy body type of Alzheimer's disease.

In another aspect, this method of treatment can treat an existingdisease, such as those listed above.

In another aspect, this method of treatment can prevent a disease, suchas those listed above, from developing or progressing.

In a preferred aspect of the methods of the invention, the subject ishuman.

The methods of the invention employ therapeutically effective amounts:for oral administration from about 0.1 mg/day to about 1,000 mg/day; forparenteral, sublingual, intranasal, intrathecal administration fromabout 0.5 to about 100 mg/day; for depo administration and implants fromabout 0.5 mg/day to about 50 mg/day; for topical administration fromabout 0.5 mg/day to about 200 mg/day; for rectal administration fromabout 0.5 mg to about 500 mg.

In a preferred aspect, the therapeutically effective amounts for oraladministration is from about 1 mg/day to about 100 mg/day; and forparenteral administration from about 5 to about 50 mg daily.

In a more preferred aspect, the therapeutically effective amounts fororal administration is from about 5 mg/day to about 50 mg/day.

The present invention also includes the use of a compound of formula(I), or a pharmaceutically acceptable salt thereof for the manufactureof a medicament for use in treating a subject who has, or in preventinga subject from developing, a disease or condition selected from thegroup consisting of Alzheimer's disease, for helping prevent or delaythe onset of Alzheimer's disease, for treating subjects with mildcognitive impairment (MCI) and preventing or delaying the onset ofAlzheimer's disease in those who would progress from MCI to AD, fortreating Down's syndrome, for treating humans who have HereditaryCerebral Hemorrhage with Amyloidosis of the Dutch-Type, for treatingcerebral amyloid angiopathy and preventing its potential consequences,i.e. single and recurrent lobar hemorrhages, for treating otherdegenerative dementias, including dementias of mixed vascular anddegenerative origin, dementia associated with Parkinson's disease,frontotemporal dementias with parkinsonism (FTDP), dementia associatedwith progressive supranuclear palsy, dementia associated with corticalbasal degeneration, diffuse Lewy body type of Alzheimer's disease andwho is in need of such treatment.

In one aspect, this use of a compound of formula (I) can be employedwhere the disease is Alzheimer's disease.

In another aspect, this use of a compound of formula (I) can helpprevent or delay the onset of Alzheimer's disease.

In another aspect, this use of a compound of formula (I) can help slowthe progression of Alzheimer's disease.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease is mild cognitive impairment.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease is Down's syndrome.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease is Hereditary Cerebral Hemorrhage with Amyloidosis ofthe Dutch-Type.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease is cerebral amyloid angiopathy.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease is degenerative dementias.

In another aspect, this use of a compound of formula (I) can be employedwhere the disease is diffuse Lewy body type of Alzheimer's disease.

In a preferred aspect, this use of a compound of formula (I) is apharmaceutically acceptable salt of an acid selected from the groupconsisting of acids hydrochloric, hydrobromic, hydroiodic, nitric,sulfuric, phosphoric, citric, methanesulfonic, CH₃—(CH₂)_(n)—COOH wheren is 0 thru 4, HOOC—(CH₂)_(n)—COOH where n is as defined above,HOOC—CH═CH—COOH, and phenyl-COOH.

The present invention also includes methods for inhibitingbeta-secretase activity, for inhibiting cleavage of amyloid precursorprotein (APP), in a reaction mixture, at a site between Met596 andAsp597, numbered for the APP-695 amino acid isotype, or at acorresponding site of an isotype or mutant thereof; for inhibitingproduction of amyloid beta peptide (A beta) in a cell; for inhibitingthe production of beta-amyloid plaque in an animal; and for treating orpreventing a disease characterized by beta-amyloid deposits in thebrain. These methods each include administration of a therapeuticallyeffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof.

The present invention also includes a method for inhibitingbeta-secretase activity, including exposing said beta-secretase to aneffective inhibitory amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

In one aspect, this method includes exposing said beta-secretase to saidcompound in vitro.

In another aspect, this method includes exposing said beta-secretase tosaid compound in a cell.

In another aspect, this method includes exposing said beta-secretase tosaid compound in a cell in an animal.

In another aspect, this method includes exposing said beta-secretase tosaid compound in a human.

The present invention also includes a method for inhibiting cleavage ofamyloid precursor protein (APP), in a reaction mixture, at a sitebetween Met596 and Asp597, numbered for the APP-695 amino acid isotype;or at a corresponding site of an isotype or mutant thereof, includingexposing said reaction mixture to an effective inhibitory amount of acompound of formula (I), or a pharmaceutically acceptable salt thereof.

In one aspect, this method employs a cleavage site: between Met652 andAsp653, numbered for the APP-751 isotype; between Met 671 and Asp 672,numbered for the APP-770 isotype; between Leu596 and Asp597 of theAPP-695 Swedish Mutation; between Leu652 and Asp653 of the APP-751Swedish Mutation; or between Leu671 and Asp672 of the APP-770 SwedishMutation.

In another aspect, this method exposes said reaction mixture in vitro.

In another aspect, this method exposes said reaction mixture in a cell.

In another aspect, this method exposes said reaction mixture in ananimal cell.

In another aspect, this method exposes said reaction mixture in a humancell.

The present invention also includes a method for inhibiting productionof amyloid beta peptide (A beta) in a cell, including administering tosaid cell an effective inhibitory amount of a compound of formula (I),or a pharmaceutically acceptable salt thereof.

In an embodiment, this method includes administering to an animal.

In an embodiment, this method includes administering to a human.

The present invention also includes a method for inhibiting theproduction of beta-amyloid plaque in an animal, including administeringto said animal an effective inhibitory amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof.

In one embodiment of this aspect, this method includes administering toa human.

The present invention also includes a method for treating or preventinga disease characterized by beta-amyloid deposits in the brain includingadministering to a subject an effective therapeutic amount of a compoundof formula (I), or a pharmaceutically acceptable salt thereof.

In one aspect, this method employs a compound at a therapeutic amount inthe range of from about 0.1 to about 1000 mg/day.

In another aspect, this method employs a compound at a therapeuticamount in the range of from about 15 to about 1500 mg/day.

In another aspect, this method employs a compound at a therapeuticamount in the range of from about 1 to about 100 mg/day.

In another aspect, this method employs a compound at a therapeuticamount in the range of from about 5 to about 50 mg/day.

In another aspect, this method can be used where said disease isAlzheimer's disease.

In another aspect, this method can be used where said disease is MildCognitive Impairment, Down's Syndrome, or Hereditary Cerebral Hemorrhagewith Amyloidosis of the Dutch Type.

The present invention also includes a composition includingbeta-secretase complexed with a compound of formula (I), or apharmaceutically acceptable salt thereof.

The present invention also includes a method for producing abeta-secretase complex including exposing beta-secretase to a compoundof formula (I), or a pharmaceutically acceptable salt thereof, in areaction mixture under conditions suitable for the production of saidcomplex.

In an embodiment, this method employs exposing in vitro.

In an embodiment, this method employs a reaction mixture that is a cell.

The present invention also includes a component kit including componentparts capable of being assembled, in which at least one component partincludes a compound of formula (I) enclosed in a container.

In an embodiment, this component kit includes lyophilized compound, andat least one further component part includes a diluent.

The present invention also includes a container kit including aplurality of containers, each container including one or more unit doseof a compound of formula (I), or a pharmaceutically acceptable saltthereof.

In an embodiment, this container kit includes each container adapted fororal delivery and includes a tablet, gel, or capsule.

In an embodiment, this container kit includes each container adapted forparenteral delivery and includes a depot product, syringe, ampoule, orvial.

In an embodiment, this container kit includes each container adapted fortopical delivery and includes a patch, medipad, ointment, or cream.

The present invention also includes an agent kit including a compound offormula (I), or a pharmaceutically acceptable salt thereof; and one ormore therapeutic agents selected from the group consisting of anantioxidant, an anti-inflammatory, a gamma secretase inhibitor, aneurotrophic agent, an acetyl cholinesterase inhibitor, a statin, an Abeta peptide, and an anti-A beta antibody.

The present invention provides compounds, compositions, kits, andmethods for inhibiting beta-secretase-mediated cleavage of amyloidprecursor protein (APP). More particularly, the compounds, compositions,and methods of the invention are effective to inhibit the production ofA beta peptide and to treat or prevent any human or veterinary diseaseor condition associated with a pathological form of A beta peptide.

The compounds, compositions, and methods of the invention are useful fortreating humans who have Alzheimer's Disease (AD), for helping preventor delay the onset of AD, for treating subjects with mild cognitiveimpairment (MCI), and preventing or delaying the onset of AD in thosesubjects who would otherwise be expected to progress from MCI to AD, fortreating Down's syndrome, for treating Hereditary Cerebral Hemorrhagewith Amyloidosis of the Dutch Type, for treating cerebral beta-amyloidangiopathy and preventing its potential consequences such as single andrecurrent lobar hemorrhages, for treating other degenerative dementias,including dementias of mixed vascular and degenerative origin, fortreating dementia associated with Parkinson's disease, frontotemporaldementias with parkinsonism (FTDP), dementia associated with progressivesupranuclear palsy, dementia associated with cortical basaldegeneration, and diffuse Lewy body type AD.

The compounds of the invention possess beta-secretase inhibitoryactivity. The inhibitory activities of the compounds of the inventionare readily demonstrated, for example, using one or more of the assaysdescribed herein or known in the art.

The compounds of formula (I) can form salts when reacted with acids.Pharmaceutically acceptable salts are generally preferred over thecorresponding compounds of formula (I) since they frequently producecompounds which are usually more water soluble, stable and/or morecrystalline. Pharmaceutically acceptable salts are any salt whichretains the activity of the parent compound and does not impart anydeleterious or undesirable effect on the subject to whom it isadministered and in the context in which it is administered.Pharmaceutically acceptable salts include acid addition salts of bothinorganic and organic acids. The preferred pharmaceutically acceptablesalts include salts of the following acids acetic, aspartic,benzenesulfonic, benzoic, bicarbonic, bisulfuric, bitartaric, butyric,calcium edetate, camsylic, carbonic, chlorobenzoic, citric, edetic,edisylic, estolic, esyl, esylic, formic, fumaric, gluceptic, gluconic,glutamic, glycollylarsanilic, hexamic, hexylresorcinoic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic,lactic, lactobionic, maleic, malic, malonic, mandelic, methanesulfonic,methylnitric, methylsulfuric, mucic, muconic, napsylic, nitric, oxalic,p-nitromethanesulfonic, pamoic, pantothenic, phosphoric, monohydrogenphosphoric, dihydrogen phosphoric, phthalic, polygalactouronic,propionic, salicylic, stearic, succinic, succinic, sulfamic, sulfanilic,sulfonic, sulfuric, tannic, tartaric, teoclic and toluenesulfonic. Forother acceptable salts, see Int. J. Pharm., 33, 201-217 (1986) and J.Pharm. Sci., 66(1), 1, (1977).

The present invention provides kits, and methods for inhibitingbeta-secretase enzyme activity and A beta peptide production. Inhibitionof beta-secretase enzyme activity halts or reduces the production of Abeta from APP and reduces or eliminates the formation of beta-amyloiddeposits in the brain.

Methods of the Invention

The compounds of the invention, and pharmaceutically acceptable saltsthereof, are useful for treating humans or animals suffering from acondition characterized by a pathological form of beta-amyloid peptide,such as beta-amyloid plaques, and for helping to prevent or delay theonset of such a condition. For example, the compounds are useful fortreating Alzheimer's disease, for helping prevent or delay the onset ofAlzheimer's disease, for treating subjects with MCI (mild cognitiveimpairment) and preventing or delaying the onset of Alzheimer's diseasein those who would progress from MCI to AD, for treating Down'ssyndrome, for treating humans who have Hereditary Cerebral Hemorrhagewith Amyloidosis of the Dutch-Type, for treating cerebral amyloidangiopathy and preventing its potential consequences, i.e. single andrecurrent lobal hemorrhages, for treating other degenerative dementias,including dementias of mixed vascular and degenerative origin, dementiaassociated with Parkinson's disease, frontotemporal dementias withparkinsonism (FTDP), dementia associated with progressive supranuclearpalsy, dementia associated with cortical basal degeneration, and diffuseLewy body type Alzheimer's disease. The compounds and compositions ofthe invention are particularly useful for treating, preventing, orslowing the progression of Alzheimer's disease. When treating orpreventing these diseases, the compounds of the invention can either beused individually or in combination, as is best for the subject.

With regard to these diseases, the term “treating” means that compoundsof the invention can be used in subjects, preferably humansubjects/patients, with existing disease. The compounds of the inventionwill not necessarily cure the subject who has the disease but will delayor slow the progression or prevent further progression of the diseasethereby giving the individual a more useful life span.

The term “preventing” means that that if the compounds of the inventionare administered to those who do not now have the disease but who wouldnormally develop the disease or be at increased risk for the disease,they will not develop the disease. In addition, “preventing” alsoincludes delaying the development of the disease in an individual whowill ultimately develop the disease or would be at risk for the diseasedue to age, familial history, genetic or chromosomal abnormalities,and/or due to the presence of one or more biological markers for thedisease, such as a known genetic mutation of APP or APP cleavageproducts in brain tissues or fluids. By delaying the onset of thedisease, compounds of the invention have prevented the individual fromgetting the disease during the period in which the individual wouldnormally have gotten the disease or reduce the rate of development ofthe disease or some of its effects but for the administration ofcompounds of the invention up to the time the individual ultimately getsthe disease. Preventing also includes administration of the compounds ofthe invention to those individuals thought to have predisposition forthe disease.

In a preferred aspect, the compounds of the invention are useful forslowing the progression of disease symptoms.

In another preferred aspect, the compounds of the invention are usefulfor preventing the further progression of disease symptoms.

In treating or preventing the above diseases, the compounds of theinvention are administered in a therapeutically effective amount. Thetherapeutically effective amount will vary depending on the particularcompound used and the route of administration, as is known to thoseskilled in the art.

In treating a subject displaying any of the diagnosed above conditions aphysician may administer a compound of the invention immediately andcontinue administration indefinitely, as needed. In treating subjectswho are not diagnosed as having Alzheimer's disease, but who arebelieved to be at substantial risk for Alzheimer's disease, thephysician should preferably start treatment when the subject firstexperiences early pre-Alzheimer's symptoms such as, memory or cognitiveproblems associated with aging. In addition, there are some subjects whomay be determined to be at risk for developing Alzheimer's through thedetection of a genetic marker such as APOE4 or other biologicalindicators that are predictive for Alzheimer's disease. In thesesituations, even though the subject does not have symptoms of thedisease, administration of the compounds of the invention may be startedbefore symptoms appear, and treatment may be continued indefinitely toprevent or delay the onset of the disease.

Dosage Forms and Amounts

The compounds of the invention can be administered orally, parenterally,(IV, IM, depo-IM, SQ, and depo SQ), sublingually, intranasally(inhalation), intrathecally, topically, or rectally. Dosage forms knownto those of skill in the art are suitable for delivery of the compoundsof the invention.

Compositions are provided that contain therapeutically effective amountsof the compounds of the invention. The compounds are preferablyformulated into suitable pharmaceutical preparations such as tablets,capsules, or elixirs for oral administration or in sterile solutions orsuspensions for parenteral administration. Typically the compoundsdescribed above are formulated into pharmaceutical compositions usingtechniques and procedures well known in the art.

About 1 to 500 mg of a compound or mixture of compounds of the inventionor a physiologically acceptable salt or ester is compounded with aphysiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, flavor, etc., in a unit dosage form as calledfor by accepted pharmaceutical practice. The amount of active substancein those compositions or preparations is such that a suitable dosage inthe range indicated is obtained. The compositions are preferablyformulated in a unit dosage form, each dosage containing from about 2 toabout 100 mg, more preferably about 10 to about 30 mg of the activeingredient. The term “unit dosage from” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical excipient.

To prepare compositions, one or more compounds of the invention aremixed with a suitable pharmaceutically acceptable carrier. Upon mixingor addition of the compound(s), the resulting mixture may be a solution,suspension, emulsion, or the like. Liposomal suspensions may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known to those skilled in the art. The form of theresulting mixture depends upon a number of factors, including theintended mode of administration and the solubility of the compound inthe selected carrier or vehicle. The effective concentration issufficient for lessening or ameliorating at least one symptom of thedisease, disorder, or condition treated and may be empiricallydetermined.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. In addition, the active materials can also be mixed withother active materials that do not impair the desired action, or withmaterials that supplement the desired action, or have another action.The compounds may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with other activeingredients.

Where the compounds exhibit insufficient solubility, methods forsolubilizing may be used. Such methods are known and include, but arenot limited to, using cosolvents such as dimethylsulfoxide (DMSO), usingsurfactants such as Tweens, and dissolution in aqueous sodiumbicarbonate. Derivatives of the compounds, such as salts or prodrugs mayalso be used in formulating effective pharmaceutical compositions.

The concentration of the compound is effective for delivery of an amountupon administration that lessens or ameliorates at least one symptom ofthe disorder for which the compound is administered. Typically, thecompositions are formulated for single dosage administration.

The compounds of the invention may be prepared with carriers thatprotect them against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems. The active compound is included in thepharmaceutically acceptable carrier in an amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the subject treated. The therapeutically effective concentration maybe determined empirically by testing the compounds in known in vitro andin vivo model systems for the treated disorder.

The compounds and compositions of the invention can be enclosed inmultiple or single dose containers. The enclosed compounds andcompositions can be provided in kits, for example, including componentparts that can be assembled for use. For example, a compound inhibitorin lyophilized form and a suitable diluent may be provided as separatedcomponents for combination prior to use. A kit may include a compoundinhibitor and a second therapeutic agent for co-administration. Theinhibitor and second therapeutic agent may be provided as separatecomponent parts. A kit may include a plurality of containers, eachcontainer holding one or more unit dose of the compound of theinvention. The containers are preferably adapted for the desired mode ofadministration, including, but not limited to tablets, gel capsules,sustained-release capsules, and the like for oral administration; depotproducts, pre-filled syringes, ampoules, vials, and the like forparenteral administration; and patches, medipads, creams, and the likefor topical administration.

The concentration of active compound in the drug composition will dependon absorption, inactivation, and excretion rates of the active compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activecompound in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules, or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches, and the like can contain any ofthe following ingredients or compounds of a similar nature: a bindersuch as, but not limited to, gum tragacanth, acacia, corn starch, orgelatin; an excipient such as microcrystalline cellulose, starch, orlactose; a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a gildant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, or fruitflavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials, whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, chewing gum orthe like. A syrup may contain, in addition to the active compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings, and flavors.

The active materials can also be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent such as water for injection, saline solution, fixed oil,a naturally occurring vegetable oil such as sesame oil, coconut oil,peanut oil, cottonseed oil, and the like, or a synthetic fatty vehiclesuch as ethyl oleate, and the like, polyethylene glycol, glycerine,propylene glycol, or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid (EDTA); buffers such as acetates,citrates, and phosphates; and agents for the adjustment of tonicity suchas sodium chloride and dextrose. Parenteral preparations can be enclosedin ampoules, disposable syringes, or multiple dose vials made of glass,plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required.

Where administered intravenously, suitable carriers includephysiological saline, phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents such as glucose,polyethylene glycol, polypropyleneglycol, and mixtures thereof.Liposomal suspensions including tissue-targeted liposomes may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known for example, as described in U.S. Pat. No.4,522,811.

The active compounds may be prepared with carriers that protect thecompound against rapid elimination from the body, such as time-releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid, and the like.Methods for preparation of such formulations are known to those skilledin the art.

The compounds of the invention can be administered orally, parenterally(IV, IM, depo-IM, SQ, and depo-SQ), sublingually, intranasally(inhalation), intrathecally, topically, or rectally. Dosage forms knownto those skilled in the art are suitable for delivery of the compoundsof the invention.

Compounds of the invention may be administered enterally orparenterally. When administered orally, compounds of the invention canbe administered in usual dosage forms for oral administration as is wellknown to those skilled in the art. These dosage forms include the usualsolid unit dosage forms of tablets and capsules as well as liquid dosageforms such as solutions, suspensions, and elixirs. When the solid dosageforms are used, it is preferred that they be of the sustained releasetype so that the compounds of the invention need to be administered onlyonce or twice daily.

The oral dosage forms are administered to the subject 1, 2, 3, or 4times daily. It is preferred that the compounds of the. invention beadministered either three or fewer times, more preferably once or twicedaily. Hence, it is preferred that the compounds of the invention beadministered in oral dosage form. It is preferred that whatever oraldosage form is used, that it be designed so as to protect the compoundsof the invention from the acidic environment of the stomach. Entericcoated tablets are well known to those skilled in the art. In addition,capsules filled with small spheres each coated to protect from theacidic stomach, are also well known to those skilled in the art.

When administered orally, an administered amount therapeuticallyeffective to inhibit beta-secretase activity, to inhibit A betaproduction, to inhibit A beta deposition, or to treat or prevent AD isfrom about 0.1 mg/day to about 1,000 mg/day. It is preferred that theoral dosage is from about 1 mg/day to about 100 mg/day. It is morepreferred that the oral dosage is from about 5 mg/day to about 50mg/day. It is understood that while a subject may be started at onedose, that dose may be varied over time as the subject's conditionchanges.

Compounds of the invention may also be advantageously delivered in anano crystal dispersion formulation. Preparation of such formulations isdescribed, for example, in U.S. Pat. No. 5,145,684. Nano crystallinedispersions of HIV protease inhibitors and their method of use aredescribed in U.S. Pat. No. 6,045,829. The nano crystalline formulationstypically afford greater bioavailability of drug compounds.

The compounds of the invention can be administered parenterally, forexample, by, IV, IM, depo-IM, SC, or depo-SC. When administeredparenterally, a therapeutically effective amount of about 0.5 to about100 mg/day, preferably from about 5 to about 50 mg daily should bedelivered. When a depot formulation is used for injection once a monthor once every two weeks, the dose should be about 0. 5 mg/day to about50 mg/day, or a monthly dose of from about 15 mg to about 1,500 mg. Inpart because of the forgetfulness of the subjects with Alzheimer'sdisease, it is preferred that the parenteral dosage form be a depoformulation.

The compounds of the invention can be administered sublingually. Whengiven sublingually, the compounds of the invention should be given oneto four times daily in the amounts described above for IMadministration.

The compounds of the invention can be administered intranasally. Whengiven by this route, the appropriate dosage forms are a nasal spray ordry powder, as is known to those skilled in the art. The dosage of thecompounds of the invention for intranasal administration is the amountdescribed above for IM administration.

The compounds of the invention can be administered intrathecally. Whengiven by this route the appropriate dosage form can be a parenteraldosage form as is known to those skilled in the art. The dosage of thecompounds of the invention for intrathecal administration is the amountdescribed above for IM administration.

The compounds of the invention can be administered topically. When givenby this route, the appropriate dosage form is a cream, ointment, orpatch. Because of the amount of the compounds of the invention to beadministered, the patch is preferred. When administered topically, thedosage is from about 0.5 mg/day to about 200 mg/day. Because the amountthat can be delivered by a patch is limited, two or more patches may beused. The number and size of the patch is not important, what isimportant is that a therapeutically effective amount of the compounds ofthe invention be delivered as is known to those skilled in the art. Thecompounds of the invention can be administered rectally by suppositoryas is known to those skilled in the art. When administered bysuppository, the therapeutically effective amount is from about 0.5 mgto about 500 mg.

The compounds of the invention can be administered by implants as isknown to those skilled in the art. When administering a compound of theinvention by implant, the therapeutically effective amount is the amountdescribed above for depot administration.

The invention here is the new compounds of the invention and new methodsof using the compounds of the invention. Given a particular compound ofthe invention and a desired dosage form, one skilled in the art wouldknow how to prepare and administer the appropriate dosage form.

The compounds of the invention are used in the same manner, by the sameroutes of administration, using the same pharmaceutical dosage forms,and at the same dosing schedule as described above, for preventingdisease or treating subjects with MCI (mild cognitive impairment) andpreventing or delaying the onset of Alzheimer's disease in those whowould progress from MCI to AD, for treating or preventing Down'ssyndrome, for treating humans who have Hereditary Cerebral Hemorrhagewith Amyloidosis of the Dutch-Type, for treating cerebral amyloidangiopathy and preventing its potential consequences, i.e. single andrecurrent lobar hemorrhages, for treating other degenerative dementias,including dementias of mixed vascular and degenerative origin, dementiaassociated with Parkinson's disease, frontotemporal dementias withparkinsonism (FTDP), dementia associated with progressive supranuclearpalsy, dementia associated with cortical basal degeneration, and diffuseLewy body type of Alzheimer's disease.

The compounds of the invention can be used with each other or with otheragents used to treat or prevent the conditions listed above. Such agentsinclude gamma-secretase inhibitors, anti-amyloid vaccines andpharmaceutical agents such as donepezil hydrochloride (ARICEPT®Tablets), tacrine hydrochloride (COGNEX® Capsules) or otheracetylcholine esterase inhibitors and with direct or indirectneurotropicagents of the future.

In addition, the compounds of the invention can also be used withinhibitors of P-glycoproten (P-gp). The use of P-gp inhibitors is knownto those skilled in the art. See for example, Cancer Research, 53,4595-4602 (1993), Clin. Cancer Res., 2, 7-12 (1996), Cancer Research,56, 4171-4179 (1996), International Publications WO99/64001 andWO01/10387. The important thing is that the blood level of the P-gpinhibitor be such that it exerts its effect in inhibiting P-gp fromdecreasing brain blood levels of the compounds of the invention. To thatend the P-gp inhibitor and the compounds of the invention can beadministered at the same time, by the same or different route ofadministration, or at different times. The important thing is not thetime of administration but having an effective blood level of the P-gpinhibitor.

Suitable P-gp inhibitors include cyclosporin A, verapamil, tamoxifen,quinidine, Vitamin E-TGPS, ritonavir, megestrol acetate, progesterone,rapamycin, 10, 11-methanodibenzosuberane, phenothiazines, acridinederivatives such as GF120918, FK506, VX-710, LY335979, PSC-833, GF-102,918 and other steroids. It is to be understood that additional agentswill be found that do the same function and are also considered to beuseful.

The P-gp inhibitors can be administered orally, parenterally, (IV, IM,IM-depo, SQ, SQ-depo), topically, sublingually, rectally, intranasally,intrathecally and by implant.

The therapeutically effective amount of the P-gp inhibitors is fromabout 0.1 to about 300 mg/kg/day, preferably about 0.1 to about 150mg/kg daily. It is understood that while a subject may be started on onedose, that dose may have to be varied over time as the subject'scondition changes.

When administered orally, the P-gp inhibitors can be administered inusual dosage forms for oral administration as is known to those skilledin the art. These dosage forms include the usual solid unit dosage formsof tablets and capsules as well as liquid dosage forms such assolutions, suspensions and elixirs. When the solid dosage forms areused, it is preferred that they be of the sustained release type so thatthe P-gp inhibitors need to be administered only once or twice daily.The oral dosage forms are administered to the subject one thru fourtimes daily. It is preferred that the P-gp inhibitors be administeredeither three or fewer times a day, more preferably once or twice daily.Hence, it is preferred that the P-gp inhibitors be administered in soliddosage form and further it is preferred that the solid dosage form be asustained release form which permits once or twice daily dosing. It ispreferred that what ever dosage form is used, that it be designed so asto protect the P-gp inhibitors from the acidic environment of thestomach. Enteric coated tablets are well known to those skilled in theart. In addition, capsules filled with small spheres each coated toprotect from the acidic stomach, are also well known to those skilled inthe art.

In addition, the P-gp inhibitors can be administered parenterally. Whenadministered parenterally they can be administered IV, IM, depo-IM, SQor depo-SQ. The P-gp inhibitors can be given sublingually. When givensublingually, the P-gp inhibitors should be given one thru four timesdaily in the same amount as for IM administration.

The P-gp inhibitors can be given intranasally. When given by this routeof administration, the appropriate dosage forms are a nasal spray or drypowder as is known to those skilled in the art. The dosage of the P-gpinhibitors for intranasal administration is the same as for IMadministration.

The P-gp inhibitors can be given intrathecally. When given by this routeof administration the appropriate dosage form can be a parenteral dosageform as is known to those skilled in the art.

The P-gp inhibitors can be given topically. When given by this route ofadministration, the appropriate dosage form is a cream, ointment orpatch. Because of the amount of the P-gp inhibitors needed to beadministered the path is preferred. However, the amount that can bedelivered by a patch is limited. Therefore, two or more patches may berequired. The number and size of the patch is not important, what isimportant is that a therapeutically effective amount of the P-gpinhibitors be delivered as is known to those skilled in the art. TheP-gp inhibitors can be administered rectally by suppository as is knownto those skilled in the art.

The P-gp inhibitors can be administered by implants as is known to thoseskilled in the art.

There is nothing novel about the route of administration or the dosageforms for administering the P-gp inhibitors. Given a particular P-gpinhibitor, and a desired dosage form, one skilled in the art would knowhow to prepare the appropriate dosage form for the P-gp inhibitor.

The compounds employed in the methods of the invention can be used incombination, with each other or with other therapeutic agents orapproaches used to treat or prevent the conditions listed above. Suchagents or approaches include: acetylcholine esterase inhibitors such astacrine (tetrahydroaminoacridine, marketed as COGNEX®), donepezilhydrochloride, (marketed as Aricept® and rivastigmine (marketed asExelon®); gamma-secretase inhibitors; anti-inflammatory agents such ascyclooxygenase II inhibitors; anti-oxidants such as Vitamin E andginkolides; immunological approaches, such as, for example, immunizationwith A beta peptide or administration of anti-A beta peptide antibodies;statins; and direct or indirect neurotropic agents such asCerebrolysin®, AIT-082 (Emilieu, 2000, Arch. Neurol. 57:454), and otherneurotropic agents of the future.

It should be apparent to one skilled in the art that the exact dosageand frequency of administration will depend on the particular compoundsemployed in the methods of the invention administered, the particularcondition being treated, the severity of the condition being treated,the age, weight, general physical condition of the particular subject,and other medication the individual may be taking as is well known toadministering physicians who are skilled in this art.

Inhibition of APP Cleavage

The compounds of the invention inhibit cleavage of APP between Met595and Asp596 numbered for the APP695 isoform, or a mutant thereof, or at acorresponding site of a different isoform, such as APP751 or APP770, ora mutant thereof (sometimes referred to as the “beta secretase site”).While not wishing to be bound by a particular theory, inhibition ofbeta-secretase activity is thought to inhibit production of beta amyloidpeptide (A beta). Inhibitory activity is demonstrated in one of avariety of inhibition assays, whereby cleavage of an APP substrate inthe presence of a beta-secretase enzyme is analyzed in the presence ofthe inhibitory compound, under conditions normally sufficient to resultin cleavage at the beta-secretase cleavage site. Reduction of APPcleavage at the beta-secretase cleavage site compared with an untreatedor inactive control is correlated with inhibitory activity. Assaysystems that can be used to demonstrate efficacy of the compoundinhibitors of the invention are known. Representative assay systems aredescribed, for example, in U.S. Pat. Nos. 5,942,400, 5,744,346, as wellas in the Examples below.

The enzymatic activity of beta-secretase and the production of A betacan be analyzed in vitro or in vivo, using natural, mutated, and/orsynthetic APP substrates, natural, mutated, and/or synthetic enzyme, andthe test compound. The analysis may involve primary or secondary cellsexpressing native, mutant, and/or synthetic APP and enzyme, animalmodels expressing native APP and enzyme, or may utilize transgenicanimal models expressing the substrate and enzyme. Detection ofenzymatic activity can be by analysis of one or more of the cleavageproducts, for example, by immunoassay, fluorometric or chromogenicassay, HPLC, or other means of detection. Inhibitory compounds aredetermined as those having the ability to decrease the amount ofbeta-secretase cleavage product produced in comparison to a control,where beta-secretase mediated cleavage in the reaction system isobserved and measured in the absence of inhibitory compounds.

Beta-Secretase

Various forms of beta-secretase enzyme are known, and are available anduseful for assay of enzyme activity and inhibition of enzyme activity.These include native, recombinant, and synthetic forms of the enzyme.Human beta-secretase is known as Beta Site APP Cleaving Enzyme (BACE),Asp2, and memapsin 2, and has been characterized, for example, in U.S.Pat. No. 5,744,346 and published PCT patent applications WO98/22597,WO00/03819, WO01/23533, and WO00/17369, as well as in literaturepublications (Hussain et al., 1999, Mol. Cell. Neurosci. 14:419-427;Vassar et al., 1999, Science 286:735-741; Yan et al., 1999, Nature402:533-537; Sinha et al., 1999, Nature 40:537-540; and Lin et al.,2000, PNAS USA 97:1456-1460). Synthetic forms of the enzyme have alsobeen described (WO98/22597 and WO00/17369). Beta-secretase can beextracted and purified from human brain tissue and can be produced incells, for example mammalian cells expressing recombinant enzyme.

Preferred methods employ compounds that are effective to inhibit 50% ofbeta-secretase enzymatic activity at a concentration of less than about50 micromolar, preferably at a concentration of less than about 10micromolar, more preferably less than about 1 micromolar, and mostpreferably less than about 10 nanomolar.

APP Substrate

Assays that demonstrate inhibition of beta-secretase-mediated cleavageof APP can utilize any of the known forms of APP, including the 695amino acid “normal” isotype described by Kang et al., 1987, Nature325:733-6, the 770 amino acid isotype described by Kitaguchi et. al.,1981, Nature 331:530-532, and variants such as the Swedish Mutation(KM670-1NL) (APP-SW), the London Mutation (V7176F), and others. See, forexample, U.S. Pat. No. 5,766,846 and also Hardy, 1992, Nature Genet.1:233-234, for a review of known variant mutations. Additional usefulsubstrates include the dibasic amino acid modification, APP-KKdisclosed, for example, in WO 00/17369, fragments of APP, and syntheticpeptides containing the beta-secretase cleavage site, wild type (WT) ormutated form, e.g., SW, as described, for example, in U.S. Pat. No5,942,400 and WO00/03819.

The APP substrate contains the beta-secretase cleavage site of APP(KM-DA or NL-DA) for example, a complete APP peptide or variant, an APPfragment, a recombinant or synthetic APP, or a fusion peptide.Preferably, the fusion peptide includes the beta-secretase cleavage sitefused to a peptide having a moiety useful for enzymatic assay, forexample, having isolation and/or detection properties. A useful moietymay be an antigenic epitope for antibody binding, a label or otherdetection moiety, a binding substrate, and the like.

Antibodies

Products characteristic of APP cleavage can be measured by immunoassayusing various antibodies, as described, for example, in Pirttila et al.,1999, Neuro. Lett. 249:21-4, and in U.S. Pat. No. 5,612,486. Usefulantibodies to detect A beta include, for example, the monoclonalantibody 6E10 (Senetek, St. Louis, Mo.) that specifically recognizes anepitope on amino acids 1-16 of the A beta peptide; antibodies 162 and164 (New York State Institute for Basic Research, Staten Island, N.Y.)that are specific for human A beta 1-40 and 1-42, respectively; andantibodies that recognize the junction region of beta-amyloid peptide,the site between residues 16 and 17, as described in U.S. Pat. No.5,593,846. Antibodies raised against a synthetic peptide of residues 591to 596 of APP and SW192 antibody raised against 590-596 of the Swedishmutation are also useful in immunoassay of APP and its cleavageproducts, as described in U.S. Pat. Nos. 5,604,102 and 5,721,130.

Assay Systems

Assays for determining APP cleavage at the beta-secretase cleavage siteare well known in the art. Exemplary assays, are described, for example,in U.S. Pat. Nos. 5,744,346 and 5,942,400, and described in the Examplesbelow.

Cell Free Assays

Exemplary assays that can be used to demonstrate the inhibitory activityof the compounds of the invention are described, for example, inWO00/17369, WO 00/03819, and U.S. Pat. Nos. 5,942,400 and 5,744,346.Such assays can be performed in cell-free incubations or in cellularincubations using cells expressing a beta-secretase and an APP substratehaving a beta-secretase cleavage site.

An APP substrate containing the beta-secretase cleavage site of APP, forexample, a complete APP or variant, an APP fragment, or a recombinant orsynthetic APP substrate containing the amino acid sequence: KM-DA orNL-DA, is incubated in the presence of beta-secretase enzyme, a fragmentthereof, or a synthetic or recombinant polypeptide variant havingbeta-secretase activity and effective to cleave the beta-secretasecleavage site of APP, under incubation conditions suitable for thecleavage activity of the enzyme. Suitable substrates optionally includederivatives that may be fusion proteins or peptides that contain thesubstrate peptide and a modification useful to facilitate thepurification or detection of the peptide or its beta-secretase cleavageproducts. Useful modifications include the insertion of a knownantigenic epitope for antibody binding; the linking of a label ordetectable moiety, the linking of a binding substrate, and the like.

Suitable incubation conditions for a cell-free in vitro assay include,for example: approximately 200 nanomolar to 10 micromolar substrate,approximately 10 to 200 picomolar enzyme, and approximately 0.1nanomolar to 10 micromolar inhibitor compound, in aqueous solution, atan approximate pH of 4 -7, at approximately 37 degrees C., for a timeperiod of approximately 10 minutes to 3 hours. These incubationconditions are exemplary only, and can be varied as required for theparticular assay components and/or desired measurement system.Optimization of the incubation conditions for the particular assaycomponents should account for the specific beta-secretase enzyme usedand its pH optimum, any additional enzymes and/or markers that might beused in the assay, and the like. Such optimization is routine and willnot require undue experimentation.

One useful assay utilizes a fusion peptide having maltose bindingprotein (MBP) fused to the C-terminal 125 amino acids of APP-SW. The MBPportion is captured on an assay substrate by anti-MBP capture antibody.Incubation of the captured fusion protein in the presence ofbeta-secretase results in cleavage of the substrate at thebeta-secretase cleavage site. Analysis of the cleavage activity can be,for example, by immunoassay of cleavage products. One such immunoassaydetects a unique epitope exposed at the carboxy terminus of the cleavedfusion protein, for example, using the antibody SW192. This assay isdescribed, for example, in U.S. Pat. No 5,942,400.

Cellular Assay

Numerous cell-based assays can be used to analyze beta-secretaseactivity and/or processing of APP to release A beta. Contact of an APPsubstrate with a beta-secretase enzyme within the cell and in thepresence or absence of a compound inhibitor of the invention can be usedto demonstrate beta-secretase inhibitory activity of the compound.Preferably, assay in the presence of a useful inhibitory compoundprovides at least about 30%, most preferably at least about 50%inhibition of the enzymatic activity, as compared with a non-inhibitedcontrol.

In one embodiment, cells that naturally express beta-secretase are used.Alternatively, cells are modified to express a recombinantbeta-secretase or synthetic variant enzyme as discussed above. The APPsubstrate may be added to the culture medium and is preferably expressedin the cells. Cells that naturally express APP, variant or mutant formsof APP, or cells transformed to express an isoform of APP, mutant orvariant APP, recombinant or synthetic APP, APP fragment, or syntheticAPP peptide or fusion protein containing the beta-secretase APP cleavagesite can be used, provided that the expressed APP is permitted tocontact the enzyme and enzymatic cleavage activity can be analyzed.

Human cell lines that normally process A beta from APP provide a usefulmeans to assay inhibitory activities of the compounds of the invention.Production and release of A beta and/or other cleavage products into theculture medium can be measured, for example by immunoassay, such asWestern blot or enzyme-linked immunoassay (EIA) such as by ELISA.

Cells expressing an APP substrate and an active beta-secretase can beincubated in the presence of a compound inhibitor to demonstrateinhibition of enzymatic activity as compared with a control. Activity ofbeta-secretase can be measured by analysis of one or more cleavageproducts of the APP substrate. For example, inhibition of beta-secretaseactivity against the substrate APP would be expected to decrease releaseof specific beta-secretase induced APP cleavage products such as A beta.

Although both neural and non-neural cells process and release A beta,levels of endogenous beta-secretase activity are low and often difficultto detect by EIA. The use of cell types known to have enhancedbeta-secretase activity, enhanced processing of APP to A beta, and/orenhanced production of A beta are therefore preferred. For example,transfection of cells with the Swedish Mutant form of APP (APP-SW); withAPP-KK; or with APP-SW-KK provides cells having enhanced beta-secretaseactivity and producing amounts of A beta that can be readily measured.

In such assays, for example, the cells expressing APP and beta-secretaseare incubated in a culture medium under conditions suitable forbeta-secretase enzymatic activity at its cleavage site on the APPsubstrate. On exposure of the cells to the compound inhibitor, theamount of A beta released into the medium and/or the amount of CTF99fragments of APP in the cell lysates is reduced as compared with thecontrol. The cleavage products of APP can be analyzed, for example, byimmune reactions with specific antibodies, as discussed above.

Preferred cells for analysis of beta-secretase activity include primaryhuman neuronal cells, primary transgenic animal neuronal cells where thetransgene is APP, and other cells such as those of a stable 293 cellline expressing APP, for example, APP-SW.

In vivo Assays: Animal Models

Various animal models can be used to analyze beta-secretase activity and/or processing of APP to release A beta, as described above. Forexample, transgenic animals expressing APP substrate and beta-secretaseenzyme can be used to demonstrate inhibitory activity of the compoundsof the invention. Certain transgenic animal models have been described,for example, in U.S. Pat. Nos.: 5,877,399; 5,612,486; 5,387,742;5,720,936; 5,850,003; 5,877,015, and 5,811,633, and in Ganes et al.,1995, Nature 373:523. Preferred are animals that exhibit characteristicsassociated with the pathophysiology of AD. Administration of thecompound inhibitors of the invention to the transgenic mice describedherein provides an alternative method for demonstrating the inhibitoryactivity of the compounds. Administration of the compounds in apharmaceutically effective carrier and via an administrative route thatreaches the target tissue in an appropriate therapeutic amount is alsopreferred.

Inhibition of beta-secretase mediated cleavage of APP at thebeta-secretase cleavage site and of A beta release can be analyzed inthese animals by measure of cleavage fragments in the animal's bodyfluids such as cerebral fluid or tissues. Analysis of brain tissues forA beta deposits or plaques is preferred.

On contacting an APP substrate with a beta-secretase enzyme in thepresence of an inhibitory compound of the invention and under conditionssufficient to permit enzymatic mediated cleavage of APP and/or releaseof A beta from the substrate, the compounds of the invention areeffective to reduce beta-secretase-mediated cleavage of APP at thebeta-secretase cleavage site and/or effective to reduce released amountsof A beta. Where such contacting is the administration of the inhibitorycompounds of the invention to an animal model, for example, as describedabove, the compounds are effective to reduce A beta deposition in braintissues of the animal, and to reduce the number and/or size of betaamyloid plaques. Where such administration is to a human subject, thecompounds are effective to inhibit or slow the progression of diseasecharacterized by enhanced amounts of A beta, to slow the progression ofAD in the, and/or to prevent onset or development of AD in a subject atrisk for the disease.

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by one of skill in the artto which this invention belongs. All patents and publications referredto herein are hereby incorporated by reference for all purposes.

Definitions

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by one of skill in the artto which this invention belongs.

All patents and publications referred to herein are hereby incorporatedby reference for all purposes.

APP, amyloid precursor protein, is defined as any APP polypeptide,including APP variants, mutations, and isoforms, for example, asdisclosed in U.S. Pat. No. 5,766,846.

A beta, amyloid beta peptide, is defined as any peptide resulting frombeta-secretase mediated cleavage of APP, including peptides of 39, 40,41, 42, and 43 amino acids, and extending from the beta-secretasecleavage site to amino acids 39, 40, 41, 42, or 43.

Beta-secretase (BACE1, Asp2, Memapsin 2) is an aspartya protease thatmediates cleavage of APP at the amino-terminal edge of A beta. Humanbeta-secretase is described, for example, in WO00/17369.

Pharmaceutically acceptable refers to those properties and/or substancesthat are acceptable to the subject from a pharmaco-logical/toxicologicalpoint of view and to the manufacturing pharmaceutical chemist from aphysical/chemical point of view regarding composition, formulation,stability, subject acceptance and bioavailability.

A therapeutically effective amount is defined as an amount effective toreduce or lessen at least one symptom of the disease being treated or toreduce or delay onset of one or more clinical markers or symptoms of thedisease.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

As noted above, depending on whether asymmetric carbon atoms arepresent, the compounds of the invention can be present as mixtures ofisomers, especially as racemates, or in the form of pure isomers,especially optical antipodes.

Salts of compounds having salt-forming groups are especially acidaddition salts, salts with bases or, where several salt-forming groupsare present, can also be mixed salts or internal salts.

Salts are especially the pharmaceutically acceptable or non-toxic saltsof compounds of formula I.

Such salts are formed, for example, by compounds of formula I: having anacid group, for example a carboxy group or a sulfo group, and are, forexample, salts thereof with suitable bases, such as non-toxic metalsalts derived from metals of groups Ia, Ib, IIa and IIb of the PeriodicTable of the Elements, for example alkali metal salts, especiallylithium, sodium or potassium salts, or alkaline earth metal salts, forexample magnesium or calcium salts, also zinc salts or ammonium salts,as well as salts formed with organic amines, such as unsubstituted orhydroxy-substituted mono-, di- or tri-alkylamines, especially mono-, di-or tri-lower alkylamines, or with quaternary ammonium bases, for examplewith methyl-, ethyl-, diethyl- or triethyl-amine, mono-, bis- ortris-(2-hydroxy-lower alkyl)-amines, such as ethanol-, diethanol- ortriethanol-amine, tris(hydroxymethyl)methylamine or2-hydroxy-tertbutylamine, N,N-di-lower alkyl-N-(hydroxy-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)-amine, orN-methyl-D-glucamine, or quaternary ammonium hydroxides, such astetrabutylammonium hydroxide. The compounds of formula I having a basicgroup, for example an amino group, can form acid addition salts, forexample with suitable inorganic acids, for example hydrohalic acids,such as hydrochloric acid or hydrobromic acid, or sulfuric acid withreplacement of one or both protons, phosphoric acid with replacement ofone or more protons, e.g. orthophosphoric acid or metaphosphoric acid,or pyrophosphoric acid with replacement of one or more protons, or withorganic carboxylic, sulfonic, sulfo or phosphonic acids or N-substitutedsulfamic acids, for example acetic acid, propionic acid, glycolic acid,succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid,fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid,glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid,2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinicacid, as well as with amino acids, such as the .alpha.-amino acidsmentioned hereinbefore, and with methanesulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, ethane-l, 2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenenesulfonic acid,naphthalene-2-sulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, or N-cyclohexylsulfamic acid (forming cyclamates)or with other acidic organic compounds, such as ascorbic acid. Compoundsof formula I having acid and basic groups can also form internal salts.

For isolation and purification purposes it is also possible to usepharmaceutically unacceptable salts.

Synthesis of Compounds

The compounds useful in the methods of the invention can be synthesizedusing the procedures set forth in Thompson, et al., J. Med. Chem., 1994;37:3100-3107. Those skilled in the art will appreciate that minormodifications, e.g., the use of protecting groups where necessary, maybe made to the specific procedures in that article to arrive atcompounds useful in the invention.

Table I illustrates some examples of the compounds useful in the methodsof this invention.

TABLE I (I)

R₁ R₂ X Cbz-Val i-propyl C═O Boc H C═O Boc Methyl C═O Boc Ethyl C═O Bocn-propyl C═O Boc i-propyl C═O Boc C(CH₃)₂CHCH₂ C═O Boc Methyl (CH₂)OHBoc i-propyl (CH₂)OH

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXAMPLES Example A

Enzyme Inhibition Assay

The compounds of the invention are analyzed for inhibitory activity byuse of the MBP-C125 assay. This assay determines the relative inhibitionof beta-secretase cleavage of a model APP substrate, MBP-C125SW, by thecompounds assayed as compared with an untreated control. A detaileddescription of the assay parameters can be found, for example, in U.S.Pat. No. 5,942,400. Briefly, the substrate is a fusion peptide formed ofmaltose binding protein (MBP) and the carboxy terminal 125 amino acidsof APP-SW, the Swedish mutation. The beta-secretase enzyme is derivedfrom human brain tissue as described in Sinha et al, 1999, Nature40:537-540) or recombinantly produced as the full-length enzyme (aminoacids 1-501), and can be prepared, for example, from 293 cellsexpressing the recombinant cDNA, as described in WO00/47618.

Inhibition of the enzyme is analyzed, for example, by immunoassay of theenzyme's cleavage products. One exemplary ELISA uses an anti-MBP captureantibody that is deposited on precoated and blocked 96-well high bindingplates, followed by incubation with diluted enzyme reaction supernatant,incubation with a specific reporter antibody, for example, biotinylatedanti-SW192 reporter antibody, and further incubation withstreptavidin/alkaline phosphatase. In the assay, cleavage of the intactMBP-C₁₂₅SW fusion protein results in the generation of a truncatedamino-terminal fragment, exposing a new SW-192 antibody-positive epitopeat the carboxy terminus. Detection is effected by a fluorescentsubstrate signal on cleavage by the phosphatase. ELISA only detectscleavage following Leu 596 at the substrate's APP-SW 751 mutation site.

Specific Assay Procedure:

Compounds are diluted in a 1:1 dilution series to a six-pointconcentration curve (two wells per concentration) in one 96-plate rowper compound tested. Each of the test compounds is prepared in DMSO tomake up a 10 millimolar stock solution. The stock solution is seriallydiluted in DMSO to obtain a final compound concentration of 200micromolar at the high point of a 6-point dilution curve. Ten (10)microliters of each dilution is added to each of two wells on row C of acorresponding V-bottom plate to which 190 microliters of 52 millimolarNaOAc, 7.9% DMSO, pH 4.5 are pre-added. The NaOAc diluted compound plateis spun down to pellet precipitant and 20 microliters/well istransferred to a corresponding flat-bottom plate to which 30 microlitersof ice-cold enzyme-substrate mixture (2.5 microliters MBP-C125SWsubstrate, 0.03 microliters enzyme and 24.5 microliters ice cold 0.09%TX100 per 30 microliters) is added. The final reaction mixture of 200micromolar compound at the highest curve point is in 5% DMSO, 20millimolar NaOAc, 0.06% TX100, at pH 4.5.

Warming the plates to 37 degrees C. starts the enzyme reaction. After 90minutes at 37 degrees C., 200 microliters/well. cold specimen diluent isadded to stop the reaction and 20 microliters/well was transferred to acorresponding anti-MBP antibody coated ELISA plate for capture,containing 80 microliters/well specimen diluent. This reaction isincubated overnight at 4 degrees C. and the ELISA is developed the nextday after a 2 hour incubation with anti-192SW antibody, followed byStreptavidin-AP conjugate and fluorescent substrate. The signal is readon a fluorescent plate reader.

Relative compound inhibition potency is determined by calculating theconcentration of compound that showed a fifty percent reduction indetected signal (IC₅₀) compared to the enzyme reaction signal in thecontrol wells with no added compound.

Example B

Cell Free Inhibition Assay Utilizing a Synthetic APP Substrate

A synthetic APP substrate that can be cleaved by beta-secretase andhaving N-terminal biotin and made fluorescent by the covalent attachmentof Oregon green at the Cys residue is used to assay beta-secretaseactivity in the presence or absence of the inhibitory compounds of theinvention. Useful substrates include the following:

[SEQ ID NO: 1] Biotin-SEVNLDAEFRC[Oregon green]KK [SEQ ID NO: 2]Biotin-SEVKMDAEFRC[Oregon green]KK [SEQ ID NO: 3]Biotin-GLNIKTEEISEISYEVEFRC[Oregon green]KK [SEQ ID NO: 4] Biotin-ADRGLTTRPGSGLTNIKTEEISEVNLDAEFC[Oregon green]KK [SEQ ID NO: 5]Biotin-FVNQHLC_(OX)GSHLVEALY- LVC_(OX)GERGFFYTPKAC[Oregon green]KK

The enzyme (0.1 nanomolar) and test compounds (0.001-100 micromolar) areincubated in pre-blocked, low affinity, black plates (384 well) at 37degrees for 30 minutes. The reaction is initiated by addition of 150millimolar substrate to a final volume of 30 microliter per well. Thefinal assay conditions are: 0.001-100 micromolar compound inhibitor; 0.1molar sodium acetate (pH 4.5); 150 nanomolar substrate; 0.1 nanomolarsoluble beta-secretase; 0.001% Tween 20, and 2% DMSO. The assay mixtureis incubated for 3 hours at 37 degrees C., and the reaction isterminated by the addition of a saturating concentration of immunopurestreptavidin. After incubation with streptavidin at room temperature for15 minutes, fluorescence polarization is measured, for example, using aLJL Acqurest (Ex485 nm/Em530 nm). The activity of the beta-secretaseenzyme is detected by changes in the fluorescence polarization thatoccur when the substrate is cleaved by the enzyme. Incubation in thepresence or absence of compound inhibitor demonstrates specificinhibition of beta-secretase enzymatic cleavage of its synthetic APPsubstrate.

Example C

Beta-Secretase Inhibition: P26-P4′SW Assay

Synthetic substrates containing the beta-secretase cleavage site of APPare used to assay beta-secretase activity, using the methods described,for example, in published PCT application WO00/47618. The P26-P4′SWsubstrate is a peptide of the sequence:

[SEQ ID NO: 6] (biotin) CGGADRGLTTRPGSGLTNIKTEEISEVNLDAEFThe P26-P1 standard has the sequence:

[SEQ ID NO: 7] (biotin) CGGADRGLTTRPGSGLTNIKTEEISEVNL.

Briefly, the biotin-coupled synthetic substrates are incubated at aconcentration of from about 0 to about 200 micromolar in this assay.When testing inhibitory compounds, a substrate concentration of about1.0 micromolar is preferred. Test compounds diluted in DMSO are added tothe reaction mixture, with a final DMSO concentration of 5%. Controlsalso contain a final DMSO concentration of 5%. The concentration of betasecretase enzyme in the reaction is varied, to give productconcentrations with the linear range of the ELISA assay, about 125 to2000 picomolar, after dilution.

The reaction mixture also includes 20 millimolar sodium acetate, pH 4.5,0.06% Triton X100, and is incubated at 37 degrees C. for about 1 to 3hours. Samples are then diluted in assay buffer (for example, 145.4nanomolar sodium chloride, 9.51 millimolar sodium phosphate, 7.7millimolar sodium azide, 0.05% Triton X405, 6 g/liter bovine serumalbumin, pH 7.4) to quench the reaction, then diluted further forimmunoassay of the cleavage products.

Cleavage products can be assayed by ELISA. Diluted samples and standardsare incubated in assay plates coated with capture antibody, for example,SW192, for about 24 hours at 4 degrees C. After washing in TTBS buffer(150 millimolar sodium chloride, 25 millimolar Tris, 0.05% Tween 20, pH7.5), the samples are incubated with streptavidin-AP according to themanufacturer's instructions. After a one hour incubation at roomtemperature, the samples are washed in TTBS and incubated withfluorescent substrate solution A (31.2 g/liter2-amino-2-methyl-1-propanol, 30 mg/liter, pH 9.5). Reaction withstreptavidin-alkaline phosphate permits detection by fluorescence.Compounds that are effective inhibitors of beta-secretase activitydemonstrate reduced cleavage of the substrate as compared to a control.

Example D

Assays using Synthetic Oligopeptide-Substrates

Synthetic oligopeptides are prepared that incorporate the known cleavagesite of beta-secretase, and optionally detectable tags, such asfluorescent or chromogenic moieties. Examples of such peptides, as wellas their production and detection methods are described in U.S. Pat. No:5,942,400, herein incorporated by reference. Cleavage products can bedetected using high performance liquid chromatography, or fluorescent orchromogenic detection methods appropriate to the peptide to be detected,according to methods well known in the art.

By way of example, one such peptide has the sequence (biotin)-SEVNLDAEF[SEQ ID NO: 8], and the cleavage site is between residues 5 and 6.Another preferred substrate has the sequenceADRGLTTRPGSGLTNIKTEEISEVNLDAEF [SEQ ID NO: 91], and the cleavage site isbetween residues 26 and 27.

These synthetic APP substrates are incubated in the presence ofbeta-secretase under conditions sufficient to result in beta-secretasemediated cleavage of the substrate. Comparison of the cleavage resultsin the presence of the compound inhibitor to control results provides ameasure of the compound's inhibitory activity.

Example E

Inhibition of Beta-Secretase Activity—Cellular Assay

An exemplary assay for the analysis of inhibition of beta-secretaseactivity utilizes the human embryonic kidney cell line HEKp293 (ATCCAccession No. CRL-1573) transfected with APP751 containing the naturallyoccurring double mutation Lys651Met52 to Asn651Leu652 (numbered forAPP751), commonly called the Swedish mutation and shown to overproduce Abeta (Citron et al., 1992, Nature 360:672-674), as described in U.S.Pat. No. 5,604,102.

The cells are incubated in the presence/absence of the inhibitorycompound (diluted in DMSO) at the desired concentration, generally up to10 micrograms/ml. At the end of the treatment period, conditioned mediais analyzed for beta-secretase activity, for example, by analysis ofcleavage fragments. A beta can be analyzed by immunoassay, usingspecific detection antibodies. The enzymatic activity is measured in thepresence and absence of the compound inhibitors to demonstrate specificinhibition of beta-secretase mediated cleavage of APP substrate.

Example F

Inhibition of Beta-Secretase in Animal Models of AD

Various animal models can be used to screen for inhibition ofbeta-secretase activity. Examples of animal models useful in theinvention include, but are not limited to, mouse, guinea pig, dog, andthe like. The animals used can be wild type, transgenic, or knockoutmodels. In addition, mammalian models can express mutations in APP, suchas APP695-SW and the like described herein. Examples of transgenicnon-human mammalian models are described in U.S. Pat. Nos. 5,604,102,5,912,410 and 5,811,633.

PDAPP mice, prepared as described in Games et al., 1995, Nature373:523-527 are useful to analyze in vivo suppression of A beta releasein the presence of putative inhibitory compounds. As described in U.S.Pat. No. 6,191,166, 4 month old PDAPP mice are administered compoundformulated in vehicle, such as corn oil. The mice are dosed withcompound (1-30 mg/ml; preferably 1-10 mg/ml). After time, e.g., 3-10hours, the animals are sacrificed, and brains removed for analysis.

Transgenic animals are administered an amount of the compound inhibitorformulated in a carrier suitable for the chosen mode of administration.Control animals are untreated, treated with vehicle, or treated with aninactive compound. Administration can be acute, i.e., single dose ormultiple doses in one day, or can be chronic, i.e., dosing is repeateddaily for a period of days. Beginning at time 0, brain tissue orcerebral fluid is obtained from selected animals and analyzed for thepresence of APP cleavage peptides, including A beta, for example, byimmunoassay using specific antibodies for A beta detection. At the endof the test period, animals are sacrificed and brain tissue or cerebralfluid is analyzed for the presence of A beta and/or beta-amyloidplaques. The tissue is also analyzed for necrosis.

Animals administered the compound inhibitors of the invention candemonstrate reduced A beta in brain tissues or cerebral fluids andreduced beta amyloid plaques in brain tissue, as compared withnon-treated controls.

Example G

Inhibition of A Beta Production in Human Subjects

Subjects suffering from Alzheimer's Disease (AD) demonstrate anincreased amount of A beta in the brain. AD subjects are administered anamount of the compound inhibitor formulated in a carrier suitable forthe chosen mode of administration. Administration is repeated daily forthe duration of the test period. Beginning on day 0, cognitive andmemory tests are performed, for example, once per month.

Subjects administered the compound inhibitors can demonstrate slowing orstabilization of disease progression as analyzed by changes in one ormore of the following disease parameters: A beta present in CSF orplasma; brain or hippocampal volume; A beta deposits in the brain;amyloid plaque in the brain; and scores for cognitive and memoryfunction, as compared with control, non-treated subjects.

Example H

Prevention of A Beta Production in Subjects at Risk for AD

Subjects predisposed or at risk for developing AD are identified eitherby recognition of a familial inheritance pattern, for example, presenceof the Swedish Mutation, and/or by monitoring diagnostic parameters.Subjects identified as predisposed or at risk for developing AD areadministered an amount of the compound inhibitor formulated in a carriersuitable for the chosen mode of administration. Administration isrepeated daily for the duration of the test period. Beginning on day 0,cognitive and memory tests are performed, for example, once per month.

Subjects administered the compound inhibitors can demonstrate slowing orstabilization of disease progression as analyzed by changes in one ormore of the following disease parameters: A beta present in CSF orplasma; brain or hippocampal volume; amyloid plaque in the brain; andscores for cognitive and memory function, as compared with control,non-treated subjects.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention emcompasses all ofthe usual variations, adaptations, or modifications, as come within thescope of the following claims and its equivalents.

1. A method of treating a subject who has Alzheimer's disease, and whois in need of such treatment comprising administering a therapeuticallyeffective amount of a compound of formula (I):

wherein R₁ is a) Cbz-Val; b) Cbz-Ala; c) Cbz; or d) Boc; R₂ is a) H; b)C₁₋₆ alkyl, alkenyl, or alkynyl; or c) cycloalkyl; and X is a) C=O; orb) (CH₂)OH; or a pharmaceutically acceptable salt thereof.
 2. The methodaccording to claim 1, wherein the compound of formula (I) is selectedfrom the group consisting of:

and pharmaceutically acceptable salts thereof.
 3. The method accordingto claim 1, further comprising the administering a P-gp inhibitor, or apharmaceutically acceptable salt thereof.
 4. A method of treatmentaccording to claim 1, further comprising administration of one or moretherapeutic agents selected from the group consisting of an antioxidant,an anti-inflammatory, a gamma secretase inhibitor, a neurotrophic agent,an acetyl cholinesterase inhibitor, a statin, an A beta peptide, and ananti-A beta peptide.